Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol–water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.     

Clarification of small volume microbial suspensions in an ultrasonic standing wave

The removal of Saccharomyces cerevisiae and Escherichia coli from 2·5 ml suspensions in ultrasonic standing wave formed at 1 or 3 MHz has been characterized. The standing wave was set up by a plane transducer and reflector mounted in the vertical plane. Cells in the ultrasonic field first concentrated in vertical planes at half wavelength separations. The ultrasound was then pulsed to allow clumps of concentrated cells to sediment in a controlled way during the short 'off' intervals. Yeast removal from suspension at a concentration of 3 × 10⁹ ml⁻¹ (14% volume v/v) was 99·5% in a total time of 4·5 min. Almost total (99·5%) clarification of prokaryote ( E. coli ) suspension was achieved here for the first time in a standing wave field. The clarification of a 1·3 × 10¹¹ ml⁻¹ (16% v/v) E. coli suspension occurred over 11·5 min. The period decreased to 7 min in the presence of a polycationic flocculant, polyethyleneimine. The implications of the results for design of systems to further reduce clarification times are discussed. Removal efficiency for both S. cerevisiae and E. coli decreased with decrease in cell concentration. This concentration dependence is shown not to be simply a consequence of acoustic interaction between single cells. Flow cytometry of stained cells detected no loss of cell viability arising from the ultrasonic procedure.     

Electroacoustic fusion of millilitre volumes of cells in physiological medium

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.     

Soil microbial diversity, community structure and denitrification in a temperate riparian zone

Nitrate (NO ₃ ^– ) removal in riparian zones bordering agricultural areas occurs via plant uptake, microbial immobilisation and bacterial denitrification. Denitrification is a desirable mechanism for removal because the bacterial conversion of NO ₃ ^– to N gases permanently removes NO ₃ ^– from the watershed. A field and laboratory study was conducted in riparian soils adjacent to Carroll Creek, Ontario, Canada, to assess the spatial distribution of denitrification relative to microbial community structure and microbial functional diversity. Soil samples were collected in March, June, and August 1997 at varying soil depths and distances from the stream. Denitrification measurements made using the acetylene block technique on intact soil cores were highly variable and did not show any trends with riparian zone location. Microbial community composition and functional diversity were determined using sole carbon source utilization (SCSU) on Biolog® GN microplates. Substrate richness, evenness and diversity (Shannon index) were greatest within the riparian zone and may also have been influenced by a rhizosphere effect. A threshold relationship between denitrification and measures of microbial community structure implied minimum levels of richness, evenness and diversity were required for denitrification.     

Retroviral transduction of adherent cells in resonant acoustic fields

Ultrasound-induced cavitation has been extensively used to enhance the efficiency of nonviral-based gene delivery. Although such unique mechanical force could possibly augment the efficacy of retrovirus-mediated gene transfer, we harnessed an alternative approach, a resonant acoustic field, to facilitate the retroviral transduction rate. NIH 3T3 fibroblast cells suspended in a culture well and mixed with ecotropic retroviruses were co-treated with megahertz resonant acoustic fields (RAF). Suspended NIH 3T3 cells under RAF treatment agglomerated at acoustic nodal planes by primary radiation force within a short exposure time. These first arrived and agglomerated cells formed bands as nucleating sites for nanometer-sized ecotropic retroviruses circulated between nodal planes to attach on and thereby increased cell-virus encounters. According to the neomycin-resistant colony assay, 2-fold increment of retroviral transduction rate was obtained by exposing cells and retroviruses in the RAF for 6 min in the presence of 8 microg/mL Polybrene.     

New techniques for three-dimensional data analysis in histopathology.

This paper describes two three-dimensional (3D) analytical techniques based on 3D mathematical morphology that have been found useful in quantifying the 3D spatial distribution of S-phase cells in a tubular tumor of the human breast. One technique is based on determining the normalized radial distribution of the S-phase cells with respect to the central axis of the tumor. The other technique is a novel extension of the polyhedra of Voronoi to quantify the distribution. The Voronoi polyhedron of a given S-phase cell nucleus is that polyhedron of minimal volume defined by planes all of which are perpendicular bisectors of the vectors extending from the given cell to all other S-phase cells in the tumor. Methods are demonstrated for generating these polyhedra and for histogramming their volumes. An illustration is given of using the histogram to sort the S-phase cells according to their 3D positional relationships. Displays showing the sorted cells in 3D and their associated Voronoi polyhedra are provided.     

Fluorescein diacetate hydrolysis as an estimator of microbial biomass on coniferous needle surfaces

Estimating microbial standing crops and microbial production in natural habitats has been difficult for microbial ecologists. The present paper describes a simple spectrophotometric assay based on the hydrolysis of fluorescein diacetate which estimates well the standing crops of microbial cells on coniferous needles and twigs. A technique is also presented for correlating optical density readings with actual dry weights of microbial cells epiphytic on needles, and thus for standardizing the assay. The assay shows promise of broad applicability to other microbial habitats.     

Microbial lipid extraction from Lipomyces starkeyi using irreversible electroporation

The aim of the study was to investigate the feasibility of using irreversible electroporation (EP) as a microbial cell disruption technique to extract intracellular lipid within short time and in an eco‐friendly manner. An EP circuit was designed and fabricated to obtain 4 kV with frequency of 100 Hz of square waves. The yeast cells of Lipomyces starkeyi (L. starkeyi) were treated by EP for 2‐10 min where the distance between electrodes was maintained at 2, 4, and 6 cm. Colony forming units (CFU) were counted to observe the cell viability under the high voltage electric field. The forces of the pulsing electric field caused significant damage to the cell wall of L. starkeyi and the disruption of microbial cells was visualized by field emission scanning electron microscopic (FESEM) image. After breaking the cell wall, lipid was extracted and measured to assess the efficiency of EP over other techniques. The extent of cell inactivation was up to 95% when the electrodes were placed at the distance of 2 cm, which provided high treatment intensity (36.7 kWh m^?3). At this condition, maximum lipid (63 mg g^?1) was extracted when the biomass was treated for 10 min. During the comparison, EP could extract 31.88% lipid while the amount was 11.89% for ultrasonic and 16.8% for Fenton's reagent. The results recommend that the EP is a promising technique for lowering the time and solvent usage for lipid extraction from microbial biomass. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:838–845, 2018     

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

In long bacterial cells,the Min system can act off‐center

In many rod‐shaped bacteria, the Min system is well‐known for generating a cell‐pole to cell‐pole standing wave oscillation with a single node at mid‐cell to align cell division. In filamentous E. coli cells, the single‐node standing wave transitions into a multi‐nodal oscillation. These multi‐nodal dynamics have largely been treated simply as an interesting byproduct of artificially elongated cells. However, a recent in vivo study by Muraleedharan et al. shows how multi‐nodal Min dynamics are used to align non‐mid‐cell divisions in the elongated swarmer cells of Vibrio parahaemolyticus. The authors propose a model where the combined actions of cell‐length dependent Min dynamics, in concert with nucleoid occlusion along the cell length and regulation of FtsZ levels ensures Z ring formation and complete chromosome segregation at a single off‐center position. By limiting the number of cell division events to one per cell at an off‐center position, long swarmer cells are preserved within a multiplying population. The findings unveil an elegant mechanism of cell‐division regulation by the Min system that allows long swarmer cells to divide without the need to ‘dedifferentiate’.     

Optimisation of introducing foreign genes into egg cells and zygotes of wheat (Triticum aestivum L.) via microinjection

Summary An expeditious and highly efficient technique of microinjection has been developed with the aim of introducing exogenous DNA into egg cells and zygotes of wheat. Using a mechanical-dissection method and a novel immobilisation approach enabled us to microinject around 15 egg cells of wheat per hour. Exposing the protoplasts to a high-frequency alternating-current field for immobilisation, a significantly higher transient expression rate of the injected genes (46% and 52% for egg cells and zygotes, respectively) could be achieved than reported thus far for plant protoplasts. Whether this high transformation efficiency is due to the highfrequency electrical field applied for immobilising the protoplasts is not known. The transformation rate appeared to be a factor depending upon the time of egg cell isolation. According to the ultrastructural observations this seems to reflect a variation in competence of the egg cells during in situ development. In order to conduct studies directed towards establishing the optimal timewindow for DNA delivery into the fertilised egg cell, the time course of DNA dynamics during zygotic development has been quantified via quantitative microspectrofluorometry.Abbreviations AC alternating current - DAE days after emasculation - FDA fluorescein diacetate - HAP hours after pollination     

Development of multilayered cell‐hydrogel composites using an acoustic focusing technique

Multilayered composites, composed of mammalian cells arranged in a hydrogel, have been prepared using an acoustic focusing technique. Acoustic focusing is a simple, nonchemical technique that allows for the fast arrangement of cells in matrices where the control of cell geometry is beneficial. Breast cancer cells (MDA‐MB231) were dispersed in a 30 wt % solution of poly(ethylene glycol) diacrylate (PEGDA) of molecular weight 400 at a density of 5 × 10⁶ cells/mL of PEGDA solution. An ultrasonic field was used to organize the cells before polymerization of PEGDA. Disk‐shaped hydrogel composites, typically 1 cm in diameter and 2‐mm thick were prepared based on a PEGDA solution volume of 130 μL. At an acoustic frequency of 2.32 MHz, composites having cells positioned within concentric cylindrical shells interspersed with zones of cell‐free hydrogel were produced. The cells were located in annuli approximately 80‐μm thick and about 300 μm apart. The structure and viability of the cells within these constructs were studied using a fluorescent LIVE/DEAD assay. The viability of the cells was on the order of 50%. For the conditions used in this study, cell death was primarily attributed to exposure of cells to the PEGDA solution prior to polymerization, rather than adverse effects of polymerization or the sound field itself. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular.In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.     

Electrophoretic properties of a population of Escherichia coli cells studied by microelectrophoresis

The technique of microelectrophoresis was used to measure the rate of E. coli cell motility under the action of a constant electric field, and the electrokinetic potential (xi-potential) was calculated from the experimental data. As was shown by analysing the results for a sample from 100 cells, the property varied within a wide range which exceeded several times the error of a single measurement. This variation was associated with the different electrosurface properties of microbial cell groups and is characteristic of a cell population. The empirical distribution of the parameter was shown to be consistent with the normal law. The results of determining the electrokinetic potential may statistically be processed with a computer, and the profile of an experimental curve will characterize the physico-chemical state of cells.     

Spatial distribution of the beta2 integrin (CD11b/CD18) and L-selectin (CD62L) adherence receptors on human neutrophils by Conventional Optical Scanning Microscopy (COSM)

Receptors such as CD62L and CD11b/CD18, are transmembrane glycoproteins which regulate leukocyte adhesive phenotype. Flow cytometry (FCM) makes it possible to assess a characterization of the cell activation level by receptor quantifying, but that technique does not integrate other factors of adherence regulation, such as spatial distribution and molecular conformation. Our study consisted in exploring the main adherence receptors on Polymorphonuclear Neutrophils (PMN) that were simultaneously analyzed by FCM and Conventional Optical Scanning Microscopy (COSM). FCM analysis showed that TNFalpha induce a decrease in CD62L expression and an increase in beta2 integrins. COSM analysis distinguished three stages of cellular distribution of CD11b/CD18 within resting PMN: most of them (about 80%) had homogeneous distribution (heterogeneous spots distributed over the entire cell surface), for 10-15% of the cells, there was a crown distribution around the widest cell diameter and in less that 10% of them receptor distribution was polarized. CD62L was in the form of heterogeneous spots distributed in a circle on the surface on non-stimulated PMN. PMN stimulation by TNFalpha was associated to a randomized clustering involving both selectin and beta2 integrin. Three-dimensional analysis elicited data not shown by quantitative cytometry. For a single averaged value of the density determined by FMC, various spatial distributions of adherence receptors are found on the surface of non-stimulated PMN. The characterization of the leukocyte adhesive phenotype has to integrate adherence receptors density as well as their spatial distribution.     

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community¹. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities^2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells⁴. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells⁴.Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies^2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.     

Fractionation of cell mixtures using acoustic and laminar flow fields

A fractionation method applicable to different populations of cells in a suspension is reported. The separation was accomplished by subjecting the suspension to a resonant ultrasonic field and a laminar flow field propagating in orthogonal directions within a thin, rectangular chamber. Steady, laminar flow transports the cell suspension along the chamber, while the ultrasonic field causes the suspended cells to migrate to the mid-plane of the chamber at rates related to their size and physical properties. A thin flow splitter positioned near the outlet divides the effluent cell suspension into two product streams, thereby allowing cells that respond faster to the acoustic field to be separated from those cells that respond more slowly. Modeling of the trajectories of individual cells through the chamber shows that by altering the strength of the flow relative to that of the acoustic field, the desired fractionation can be controlled. Proof-of-concept experiments were performed using hybridoma cells and Lactobacillus rhamnosus cells. The two populations of cells could be effectively separated using this technique, resulting in hybridoma/Lactobacillus ratios in the left and right product streams, normalized to the feed ratio, of 6.9 +/- 1.8 and 0.39 +/- 0.01 (vol/vol), respectively. The acoustic method is fast, efficient, and could be operated continuously with a high degree of selectivity and yield and with low power consumption. (c) 2004 Wiley Periodicals, Inc.     

Standing calcium gradients in olfactory receptor neurons can be abolished by amiloride or ruthenium red

Digital imaging and the patch clamp technique were used to investigate the intracellular calcium concentration in olfactory receptor neurons using the Ca2+ indicator dyes fura-2 and fura-2/AM. The spatial distribution of Cai2+ as well as its modification by the drugs Amiloride and Ruthenium Red were studied. Resting calcium concentrations in cells loaded with fura-2/AM were between 10 and 200 nM. In cells that were loaded with the pentapotassium salt of fura-2 through the patch pipette, calcium concentrations were in the same range if ATP was added to the pipette solution. Otherwise, Ca2+ reached concentrations of approximately 500 nM. Most of the observed cells showed a standing gradient of calcium, the calcium concentrations in the distal dendritic end of the cell being higher than in the soma. In some cells, the gradient was markedly reduced or abolished by adding either Amiloride or Ruthenium Red to the bath solution. In a few cells, neither drug had any effect upon the gradient. It is suggested that the inhomogenous spatial distribution of intracellular calcium in olfactory cells of Xenopus laevis is brought about by an influx of calcium ions through two different calcium permeable conductances in the peripheral compartments of the cells. The fact that only either Ruthenium Red or Amiloride abolished the standing calcium gradient further suggested that the two conductances blocked were presumably not coexpressed in the same cells.     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.